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1.
Gastroenterology ; 161(4): 1229-1244.e9, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34147519

RESUMO

BACKGROUND & AIMS: The pathogenesis of immune checkpoint inhibitor (ICI)-colitis remains incompletely understood. We sought to identify key cellular drivers of ICI-colitis and their similarities to idiopathic ulcerative colitis, and to determine potential novel therapeutic targets. METHODS: We used a cross-sectional approach to study patients with ICI-colitis, those receiving ICI without the development of colitis, idiopathic ulcerative colitis, and healthy controls. A subset of patients with ICI-colitis were studied longitudinally. We applied a range of methods, including multiparameter and spectral flow cytometry, spectral immunofluorescence microscopy, targeted gene panels, and bulk and single-cell RNA sequencing. RESULTS: We demonstrate CD8+ tissue resident memory T (TRM) cells are the dominant activated T cell subset in ICI-colitis. The pattern of gastrointestinal immunopathology is distinct from ulcerative colitis at both the immune and epithelial-signaling levels. CD8+ TRM cell activation correlates with clinical and endoscopic ICI-colitis severity. Single-cell RNA sequencing analysis confirms activated CD8+ TRM cells express high levels of transcripts for checkpoint inhibitors and interferon-gamma in ICI-colitis. We demonstrate similar findings in both anti-CTLA-4/PD-1 combination therapy and in anti-PD-1 inhibitor-associated colitis. On the basis of our data, we successfully targeted this pathway in a patient with refractory ICI-colitis, using the JAK inhibitor tofacitinib. CONCLUSIONS: Interferon gamma-producing CD8+ TRM cells are a pathological hallmark of ICI-colitis and a novel target for therapy.


Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Colite/induzido quimicamente , Colo/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/efeitos adversos , Memória Imunológica/efeitos dos fármacos , Interferon gama/metabolismo , Células T de Memória/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/metabolismo , Estudos de Casos e Controles , Colite/tratamento farmacológico , Colite/imunologia , Colite/metabolismo , Colite Ulcerativa/imunologia , Colite Ulcerativa/metabolismo , Colo/imunologia , Colo/metabolismo , Estudos Transversais , Perfilação da Expressão Gênica , Humanos , Estudos Longitudinais , Ativação Linfocitária/efeitos dos fármacos , Células T de Memória/imunologia , Células T de Memória/metabolismo , Fenótipo , Piperidinas/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Estudos Prospectivos , Pirimidinas/uso terapêutico , RNA-Seq , Análise de Célula Única , Transcriptoma
2.
J Crohns Colitis ; 14(10): 1446-1461, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32179884

RESUMO

BACKGROUND AND AIMS: Lymphocyte activation gene [LAG]-3 is an immune checkpoint and its expression identifies recently activated lymphocytes that may contribute to inflammation. We investigated the role of LAG-3 by analysing its expression and function in immune cells from blood and tissue of patients with ulcerative colitis [UC]. METHODS: The phenotypic properties of LAG-3+ T cells were determined by flow cytometry, qRT-PCR and single-cell RNA-sequencing. LAG-3+ cells were quantified and correlated with disease activity. The functional effects of LAG-3+ cells were tested using a depleting anti-LAG-3 monoclonal antibody [mAb] in a mixed lymphocyte reaction [MLR]. RESULTS: LAG-3+ cells in the blood were negligible. LAG-3+ lymphocytes were markedly increased in inflamed mucosal tissue and both frequencies of LAG-3+ T cells and transcript levels of LAG3 correlated with endoscopic severity. LAG-3 expression was predominantly on effector memory T cells, and single-cell RNA-sequencing revealed LAG3 expression in activated and cytokine-producing T cell subsets. Foxp3+CD25hi Tregs also expressed LAG-3, although most mucosal Tregs were LAG-3-. Mucosal LAG-3+ cells produced mainly interferon γ [IFNγ] and interleukin-17A. LAG-3+ cell numbers decreased in patients who responded to biologics, and remained elevated in non-responders. Treatment with a depleting anti-LAG-3 mAb led to a reduction in proliferation and IFNγ production in an MLR. CONCLUSIONS: LAG-3+ cells are increased in the inflamed mucosa, predominantly on effector memory T cells with an activated phenotype and their cell numbers positively correlate with disease activity. Depleting LAG-3 eliminates activated proliferating T cells, and hence LAG-3 could be a therapeutic target in UC.


Assuntos
Antígenos CD/imunologia , Colite Ulcerativa , Mucosa Intestinal , Ativação Linfocitária/imunologia , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Desenvolvimento de Medicamentos , Endoscopia/métodos , Humanos , Proteínas de Checkpoint Imunológico/imunologia , Inflamação/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Gravidade do Paciente , Índice de Gravidade de Doença , Subpopulações de Linfócitos T , Proteína do Gene 3 de Ativação de Linfócitos
3.
Immunol Cell Biol ; 2018 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-29505094

RESUMO

Human blood monocytes are subclassified as classical, intermediate and nonclassical. In this study, it was shown that conventionally defined human intermediate monocytes can be divided into two distinct subpopulations with mid- and high-level surface expression of HLA-DR (referred to as DRmid and DRhi intermediate monocytes). These IM subpopulations were phenotypically and functionally characterized in healthy adult blood by flow cytometry, migration assays and lipoprotein uptake assays. Their absolute numbers and proportions were then compared in blood samples from obese and nonobese adults. DRmid and DRhi intermediate monocytes differentially expressed several proteins including CD62L, CD11a, CX3CR1 and CCR2. Overall, the DRmid intermediate monocytes surface profile more closely resembled that of classical monocytes while DRhi intermediate monocytes were more similar to nonclassical. However, in contrast to classical monocytes, DRmid intermediate monocytes migrated weakly to CCL2, had reduced intracellular calcium flux following CCR2 ligation and favored adherence to TNFα-activated endothelium over transmigration. In lipid uptake assays, DRmid intermediate monocytes demonstrated greater internalization of oxidized and acetylated low-density lipoprotein than DRhi intermediate monocytes. In obese compared to nonobese adults, proportions and absolute numbers of DRmid , but not DRhi intermediate monocytes, were increased in blood. The results are consistent with phenotypic and functional heterogeneity within the intermediate monocytes subset that may be of specific relevance to lipoprotein scavenging and metabolic health.

4.
Inflamm Bowel Dis ; 22(12): 2863-2878, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27824651

RESUMO

BACKGROUND: Infliximab (IFX), an anti-tumour necrosis factor alpha (TNFα) monoclonal antibody, provides clinical benefits in treating Crohn's disease (CD) but its mechanisms of action are not fully elucidated. This study investigated blood monocyte repertoires and the acute effects of IFX infusion on monocyte subset phenotype and function in IFX-treated patients with CD. METHODS: Monocytes and monocyte subsets were enumerated and phenotypically characterized by multicolor flow cytometry in freshly isolated blood from healthy controls (n = 21) and patients with CD treated with (IFX, n = 24) and without (non-IFX, n = 20) IFX. For the IFX-CD group, blood was sampled immediately before (tough-IFX) and after (peak-IFX) infusion. Monocyte responses to lipopolysaccharide were analyzed by whole-blood intracellular cytokine staining. RESULTS: Non-IFX and IFX-CD patients had increased numbers of intermediate (CD14CD16) monocytes compared with healthy controls, whereas classical (CD14CD16) and nonclassical (CD14CD16) monocytes were numerically reduced in the IFX-CD group alone. In all groups, monocyte subsets expressed high surface levels of transmembrane (tm)TNFα. After IFX infusion, a significant reduction in monocyte numbers occurred. Post-IFX monocytopenia was proportionately greatest for classical and intermediate subsets, correlated with postinfusion IFX levels and was not associated with monocyte apoptosis. In contrast, lipopolysaccharide-induced production of TNFα and IL-12 by monocytes was significantly reduced in peak-IFX compared with trough-IFX blood samples. CONCLUSIONS: Actively managed CD is associated with monocyte repertoire skewing suggestive of chronic inflammatory stimulation. Infused IFX acutely targets monocytes, likely by binding to tmTNFα, resulting in a non-apoptosis-related decline in circulating monocyte numbers and blunting of the inflammatory response of monocytes remaining in the blood.


Assuntos
Doença de Crohn/tratamento farmacológico , Fármacos Gastrointestinais/farmacologia , Infliximab/farmacologia , Monócitos/efeitos dos fármacos , Adulto , Idoso , Estudos de Casos e Controles , Doença de Crohn/sangue , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/citologia , Adulto Jovem
5.
Inflamm Bowel Dis ; 21(12): 2909-20, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26348448

RESUMO

Tumor necrosis factor alpha (TNF-α) has been widely accepted as a therapeutic target for inflammatory disorders including inflammatory bowel disease. Anti-TNF-α monoclonal antibodies (mAbs) including infliximab, adalimumab, golimumab, and certolizumab pegol have revolutionized therapy for these chronic inflammatory disorders. These agents are potent inhibitors of TNF-α, but significant evidence points to the fact that their actions extend beyond simple neutralization of the cytokine. Recent advances in understanding the mechanism of action of anti-TNF-α mAbs has discovered a number of previously unrecognized actions that are likely to be relevant in mediating their anti-inflammatory effects. Many of those actions are mediated by the binding of the antibodies to transmembrane TNF-α (tmTNF-α) and involve complex interactions with other molecular factors and cells. In this review, we have highlighted new information on the mechanism of actions of anti-TNF-α mAbs, from in vitro and in vivo studies. Despite obvious benefits in many patients, the clinical use of these antibodies are hampered by the fact that some patients do not respond to them, and among patients who do respond, many will develop recurrent disease despite continued dosing. Although pharmacokinetic factors explain some of the observed cases of partial or complete resistance to the effects of anti-TNF-α mAbs, other nonresponder patients may be resistant to those agents mechanism of action. A more thorough understanding of the mechanism of action of anti-TNF-α mAbs may allow the development of strategies to individualize therapy and to overcome resistance.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adalimumab/farmacologia , Anticorpos Monoclonais/farmacologia , Certolizumab Pegol/farmacologia , Resistência a Medicamentos/imunologia , Humanos , Doenças Inflamatórias Intestinais/imunologia , Infliximab/farmacologia
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